All documents to which reference is made herein are incorporated by reference in their entirety. βGBP is a negative cell cycle regulator (Wells V, Mallucci L 1991) with anti-cancer properties (Mallucci et al., 2003), recently identified as an anti-proliferative cytokine produced by activated CD4+ and CD8+ T cells (Blaser et al., 1998). It is also endogenously released by somatic cells. βGBP from several species has been cloned, expressed and purified. Thus, recombinant βGBP (for example human Hu-r-βGBP) is also available. All types of βGBP, irrespective of source, are simply referred to as βGBP herein.
βGBP is also known to induce programmed cell death (apoptosis) in cancer cells without harming normal cells. Thus βGBP enforces different regulatory functions in normal and cancer cells.
Cell division is a normal part of growth. It may also occur under other circumstances in a healthy individual, for example clonal expansion of lymphocytes during an immune reaction to a pathogen. However, if cell proliferation and growth is insufficiently controlled excessive cell division may occur. This excessive, or otherwise undesirable cell division may be considered to be disease associated cell division. Such cell division is a characteristic of hyperproliferative conditions.
One of the best known hyperproliferative conditions is cancer, although the term does encompass other conditions. These include self immune responses (such as autoimmune diseases) and immune responses against transplanted organs, tissues or cells (sometimes termed xeno reactions). Proliferation of activated T cells is involved in the pathology in many immune conditions mediated by undesirable cell division.
Cancer may result from a breakdown of the complex web of regulatory signals to which normal cells are subjected in healthy individuals. Under normal physiological conditions cells receive and respond to a wide range of stimuli, some favouring proliferation, some inhibiting proliferation. Similarly cells receive and respond to different stimuli that are either pro- or anti-apoptotic. In this manner a balance is preserved. Cell populations are replenished, replaced or expanded as necessary, but excessive proliferation is kept in check or apoptosis is enforced to maintain appropriate cell populations.
Owing to the highly complex nature of this regulation, and the many different effectors involved, the development of different cancer types and cancers in different individuals may result from a wide variety of changes or disturbances to the normal regulatory network.
As cancer develops, further mutations may accumulate in cancer cells, leading to greater disregulation of cellular functions. Selective pressure as the cancer cells divide tends to result in a stronger and stronger proliferative (or mitogentic) character and weaker response to anti-proliferative signals. Additionally, as cancer cells develop and become more aggressive, they may be forced to rely increasingly on survival signalling in order to overcome (and survive) an increase in the pro-apoptotic signalling which would normally remove such a cell from the population.
In addition, selective pressure brought about by the presence of chemotherapeutic agents leads to the development of drug resistance in many cancers.
In summary, cancers frequently result from an accumulation of mutations in a population of cells. The development of new mutations in individual cells and the inheritance of these mutations by new daughter cells typically results in a spectrum of cellular phenotypes and behaviours within a tumour or population of cancer cells.
These may range from less aggressive phenotypes (e.g. lower rates of division, greater sensitivity to normal physiological stimuli such as negative regulators of growth and pro-apoptopic signals), to more aggressive phenotypes, (e.g. higher rates of division, lower sensitivity (or the absence of sensitivity) to normal physiological stimuli such as negative regulators of growth and pro-apoptopic signals, reliance on survival signalling to avoid apoptosis, and increased drug resistance).
Selective pressure within such a population of cells tends to result in the development of increasing numbers of ever more aggressive cellular phenotypes as further mutations accumulate in the cells. Clones of cancer cells that are more aggressive and dividing more rapidly tend to out-compete less aggressive clones. Thus the character of the cancer overall tends to become more aggressive and therefore more serious and life threatening as time passes and further mutations accumulate.
Genetic alterations which in cancer cells magnify mitogenic signalling and are a cause of aggressive disease and resistance to therapies include amplification of the erbB2 (HER/neu) gene, present in many types of cancer and frequent in breast, ovarian and stomach carcinomas, and point mutations of the ras genes, common in about 15-20% of all tumors.
ErbB2 is a ligand-less member of the ErbB/EGF tyrosine kinase receptor family which magnifies mitogenic signalling by being constitutively active, by dimerising as a preferred partner with other ErbB members, which in breast cancer can be overexpressed, and by resisting endocytic degradation (Hynes et al., 1994, Yu and Hung 2000, Mendelsohn and Baselga 2000, Harari and Yarden 2000).
Phosphorylated tyrosine residues in the cytoplasmic tail of the ErbB2 molecule lead to the formation of high affinity binding sites for the SH2 domains of Shc and Grb2 adapter proteins (Segatto et al., 1990, Dankart et al., 1997), the binding of the nucleotide exchange factor SOS to the SH3 domains of Grb2, the conversion of GDP-Ras to active GTP-Ras and the activation of effector pathways which transduce proliferative, migratory and survival signalling (Mitin et al., 2005).
Ras proteins which harbour a single missense mutation magnify mitogenic signalling by chronically activating Raf serine/threonine kinases to force a phosphorylation cascade which enhances ERK activity and cell proliferation (Bos 1989, Downward 2003, Repasky et al., 2004). Critically, by interacting with the catalytic subunit of class IA (Rodriguez-Viciana et al., 1994) and class IB (Pacold et al., 2000, Walker et al., 2000 and Djodjevic et al., 2002) phosphatidylinositol-3-OH kinase (PI3 Kinase or PI3K), continuously activated Ras and constitutively active mutated Ras can contribute to coupling mitogenic input with survival ability.
By catalysing the conversion of phosphoinositide(4,5)P2 to phophoinositide(3,4,5)P3, PI3K allows Akt/PKB recruitment to the plasma membrane where Akt is activated. Phosphorylation of downstream targets such as Bad, caspase 9, forkhead transcription factors and IKKα by activated Akt confers resistance to apoptosis (Hennessy et al., 2005).
PI3K also regulates signalling networks that mediate cell growth (Foukas et al., 2006), cell proliferation (Wennstrom and Downward 1999), cytoskeletal organization (Rodriguez-Viciana et al., 1997), cell motility and migration (Vivanco and Sawyers 2002), all processes of central importance to the evolution of aggressive tumorigenesis.
The identification of multiple binding partners and downstream effectors of PI3K and of Ras has provided scope for the design of anticancer drugs specifically aimed at selected molecular targets (Downward 2003, Hennessy et al., 2005, Gibbs Chang et al., 2003 Luo J et al., 2003 and Baselga 2006), but questions have been raised concerning possible reasons for the as yet limited therapeutic success of these strategies. Concerns include the ability of intracellular signalling to integrate and bypass individual blocks, the possible molecular promiscuity of the targeting compound, lack of cell specificity, cytotoxicity and drug resistance (Johnstone et al., 2002, Mallucci et al., 2003 and Mallucci and Wells 2005).
Similarly to cancer, certain immune reactions may involve disease associated, i.e. undesirable cell division. For example, such cell division may occur among lymphocytes.
Autoimmunity is an example of an undesirable immune reaction which involves undesirable proliferation of lymphocytes such as T cells. Typically, these cells mediate immune reactions against endogenous targets within the individual concerned. The usual control mechanisms which should restrain activation and proliferation of such self reactive cells may for some reason break down in autoimmunity. Thus pathology results from the activities of a proliferating pool of cells targeting the self. The pool of proliferating lymphocytes in an autoimmune condition will typically comprise cells which are considered aggressive, or are dividing persistently. An example of an autoimmune condition is systemic lupus erythematosus (also known as SLE or lupus).
Alloimmunity is a condition that is related to autoimmunity. In alloimmune reactions the body gains immunity, from an exogenous source, e.g. another individual of the same species, against its own cells. Alloimmunity differs from autoimmunity since in the former condition the immune system attacks the body's own cells without being provoked or influenced by exogenous substances.
Alloimmunity may occur for example in the recipient of a transfusion of fluids such as blood or plasma, in the recipient of an allografts (grafts), or in the fetus after maternal antibodies have passed through the placenta into the fetus (e.g. in haemolytic disease of the newborn or fetomaternal alloimmune thrombocytopenia). Alloimmunity may be considered to be ‘provoked autoimmunity’.
An alternative, but mechanistically related problem arises when biological components such as organs, tissues or cells (i.e. grafts) are transplanted between genetically distinct individuals, resulting in an immune reaction against the graft. Whole organs may be transplanted, alternatively portions of organs, or quantities of bone marrow (e.g. comprising progenitor cells) may be transferred from donor to recipient. It may also occur as a result of a transfusion of fluids such as blood. As used herein the term “graft” is used to refer to all such transplants, transfusions and grafts.
Even where tissue matching is carried out, if the donor and recipient are genetically distinct, there remains a risk that the recipient's immune system will recognise the graft and mount an immune response against it. This is commonly known as transplant rejection. It may be a particular problem where transplantation of tissues, organs or cells occurs between individuals of different species (xenotransplantation) owing to the greater genetic and consequent molecular difference between donor and recipient.
It is standard procedure to reduce risk of rejection and the severity of the resultant immune response through the use of immune suppressive drugs. These commonly act to damp down the response of the immune system, but carry with them some risk of illness, for example through a reduced ability of the transplant recipient to fight off infectious diseases or opportunistic pathogens.
The immune reaction against a graft will of course generally involve T cells. T cells which are reactive against, or recognise molecular targets derived from the graft will become activated and proliferate. Under these, or similar circumstances, an otherwise normal immune reaction (i.e. to a ‘foreign’ target in the body) may cause pathology as the graft is attacked. Such rejection is naturally considered to be a disease, even though in the context of the functioning of the immune system, it is a ‘normal’ reaction to a foreign body. Thus, undesirable cell division (proliferation of lymphocytes, such as cells which target the graft) is of key importance in the development of immune reactions against transplanted organs, tissues or cells.
A further example of an immune condition in which undesirable cell division occurs is ‘Graft versus host disease’ following a bone marrow transplantation (or transfusion). T cells present in the graft, either as contaminants or intentionally introduced into the host, attack the tissues of the transplant recipient after perceiving host tissues as antigenically foreign. The T cells produce an excess of cytokines, including TNFα and interferon-gamma (IFNγ). A wide range of host antigens can initiate graft-versus-host-disease, among them the human leukocyte antigens (HLAs). However, graft-versus-host disease can occur even when HLA-identical siblings are the donors.
Hypersensitivity reactions are undesirable (damaging, discomfort-producing and sometimes fatal) reactions produced by the normal immune system. Hypersensitivity reactions require a pre-sensitized (immune) host. A four-group classification was expounded by Gell and Coombs.
Therefore undesirable cell division links many conditions of ill health, such as cancer, autoimmune disease, transplant rejection and graft versus host disease. In these conditions the body's aggressive reaction (or the reaction of the transplanted T cells) may be characterised by a rapidly dividing pool of cells (e.g. activated lymphocytes). Proliferating activated lymphocytes may be considered to be hyperproliferative within the meaning of this invention. A link between all of the conditions discussed is the persistence of proliferation, especially in the face of a variety of stimuli opposed to such proliferation.
βGBP has attracted interest as a potential therapeutic agent for a number of reasons. Importantly, βGBP is a natural regulatory molecule, produced primarily by the immune system, which because of its very nature and function, will not harm normal cells. βGBP is believed to play a part in the natural mechanisms and feedback circuitries governing the regulation of immune cell proliferation. In addition, βGBP is thought to play a part in cancer prevention and surveillance constitutively.
Low concentrations of βGBP are already known to inhibit the growth of T cells in vitro (Blaser et al., 1998). It has also been disclosed that at very much higher (i.e. micromolar) concentrations homodimers formed from βGBP (a complex with lectin-like characteristics known as Galectin-1 or GAL-1) can induce rapid apoptosis in activated T cells in vitro. This property is only evident at high concentrations where GAL-1 (i.e. the dimeric molecule) forms (Rabinovich et al., 1999).
It is however important to make the distinction between the known activities of GAL-1 and βGBP. This distinction is discussed in the prior art, for example in WO 92/07938, and summarised briefly below.
GAL-1 mediates its pro-apoptotic effects through its Lectin-like properties. High concentrations of GAL-1 rapidly induce apoptosis in activated T cells. Competition with lactose inhibits the pro-apoptotic effects of GAL-1, suggesting that they are mediated through crosslinking of cell surface glycoproteins.
In contrast the growth inhibitory effects of βGBP are independent of the carbohydrate binding site (i.e. independent of GAL-1 type lectin activity). This is demonstrated by the fact that the addition of lactose does not block βGBP activity and also that the 18000 Mr murine form of βGBP, in which the carbohydrate binding site is blocked by a glycan complex, has full growth inhibitory activity. Thus βGBP monomers (which do not have lectin-like characteristics) mediate their effects via an alternative mode of action. βGBP is believed to interact with a specific cell surface receptor and induce signal transduction events.
The threshold concentration for apoptotic activity of GAL-1 has previously been shown to be 4 μg/ml (Rabinovich et al., 1999, Rabinovich et al., 1998, Rabinovich et al., 1997). It was previously shown that the dimeric molecule was necessary for the biological effects (i.e. induction of apoptosis in T cells) of GAL-1. However, the biological activities of βGBP with regard to the inhibition of cell division, rather than the induction of apoptosis, are known to be exerted at concentrations measured in the range of nanograms per ml. Thus it is possible to make a clear distinction between the known activities and properties of βGBP (cytokine-like) and GAL-1 (lectin-like).
It would be useful if there were further therapeutic options in the treatment of conditions which involve disease associated cell division. Such disease associated cell division may alternatively be considered as ‘undesirable’ cell division since as described above the cell division may take place as part of a breakdown of normal control (e.g. cancer or other hyperproliferative conditions), or under circumstances where the division might be regarded as ‘normal’ but misdirected (e.g. lymphocyte proliferation in autoimmune conditions) or ‘normal’ but unwanted (e.g. an immune response to a foreign tissue that is beneficial such as a transplanted organ).
It is an object of the invention to provide medicaments useful in the treatment of such conditions, or to at least provide the public and/or medical community with a useful alternative.